Synthetic methods for preparing procyanidin oligomers

ABSTRACT

Processes are disclosed for the production of linear and branched procyanidin oligomers having “n” procyanidin monomeric units where n is 2 to 18. The processes include coupling protected, activated monomers with an unprotected monomer to produce a partially protected (4→8) dimer. The dimer is optionally blocked, coupled with an activated protected monomer to produce a partially protected, optionally blocked trimer, and deprotected. The steps can be repeated to produce higher oligomers. Processes are also provided for producing (8→8), (8→6), and (6→6) dimers and doubly branched oligomers. Crystalline 8-bromo tetra-O-benzyl (−)-epicatechin is produced under certain conditions.

This application is a division and continuation of Ser. No. 10/212,973filed Aug. 6, 2002, now U.S. Pat. No. 6,864,377 issued Mar. 8, 2005,which is a continuation of Ser. No. 09/292,244 filed Apr. 15, 1999, nowU.S. Pat. No. 7,015,338 issued Mar. 21, 2006.

FIELD OF INVENTION

This invention relates to synthetic procyanidin oligomers and methodsfor making and using the oligomers.

BACKGROUND OF THE INVENTION

Polyphenols are a highly diverse group of compounds (Ferreira, D.,Steynberg, J. P., Roux, D. G. and Brandt, E. V., Tetrahedron, 48 (10),1743-1803 (1992)) which widely occur in a variety of plants, some ofwhich enter into the food chain. In some cases they represent animportant class of compounds for the human diet. Although some of thepolyphenols are considered to be non-nutritive, interest in thesecompounds has increased because of their possible beneficial effects onhealth.

For instance, quercetin has been shown to possess anticarcinogenicactivity in experimental animal studies (Decshner, E. E., Ruperto, J.,Wong, G. and Newmark, H. L., Carcinogenesis, 7, 1193-1196 (1991) andKato, R., Nakadate, T., Yamamoto, S. and Sugimura, T., Carcinogenesis,4, 1301-1305 (1983)). (+)-Catechin and (−)-epicatechin have been shownto inhibit leukemia virus reverse transcriptase activity (Chu, S.-C.,Hsieh, Y.-S. and Lim, J.-Y., J. of Natural Products, 55 (2), 179-183,(1992)). Nobatanin (an oligomeric hydrolyzable tannin) has also beenshown to possess anti-tumor activity (Okuda, T., Yoshida, T., andHatano, T., Molecular Structures and Pharmacological Activities ofPolyphenols—Oligomeric Hydrolyzable Tannins and Others—Presented at theXVIth International Conference of the Group Polyphenols, Lisbon,Portugal, Jul. 13-16, 1992). Statistical reports have also shown thatstomach cancer mortality is significantly lower in the tea-producingdistricts of Japan. Epigallocatechin gallate has been reported to be thepharmacologically active material in green tea that inhibits mouse skintumors (Okuda et al., Ibid.). Ellagic acid has also been shown topossess anticarcinogen activity in various animal tumor models(Boukharta, M., Jalbert, G. and Castonguay, A., Efficacy ofEllagitannins and Ellagic Acid as Cancer Chemopreventic Agents—Presentedat the XVIth International Conference of the Group Polyphenols, Lisbon,Portugal, Jul. 13-16, 1992). Proanthocyanidin oligomers have beenpatented by the Kikkoman Corporation for use as antimutagens. The use ofphenolic compounds in foods and their modulation of tumor development inexperimental animal models has been recently presented at the 202^(nd)National Meeting of the American Chemical Society (Phenolic Compounds inFoods and Their Effects on Health I, Analysis, Occurrence & Chemistry,Ho, C.-T., Lee, C.Y. and Huang, M.-T. editors, ACS Symposium Series 506,American Chemical Society, Washington, D.C. (1992); Phenolic Compoundsin Foods and Their Effects on Health II. Antioxidants & CancerPrevention, Huang, M.-T., Ho, C.-T. and Lee, C.Y. editors, ACS SymposiumSeries 506, American Chemical Society, Washington, D.C. (1992)).

However, these citations do not relate to cocoa extracts or compoundstherefrom or to any methods for preparing such extracts or compoundstherefrom, or to any of the uses described in U.S. Pat. No. 5,554,645issued Sep. 10, 1996 to Romanczyk et al., U.S. Pat. No. 5,712,305 issuedJan. 27, 1998 to Romanczyk et al., and U.S. Pat. No. 5,650,432 issuedJul. 22, 1997 to Walker et al.

Isolation, separation, purification, and identification methods havebeen established for the recovery of a range of procyanidin oligomersfor comparative in vitro and in vivo assessment of biologicalactivities. For instance, anti-cancer activity is elicited by pentamericthrough decameric procyanidins, but not by monomers through tetramericcompounds. Currently, gram quantities of pure (>95%) pentamer areobtained by time-consuming methods. These methods are not satisfactoryfor obtaining sufficient quantities of the pentamer for large scalepharmacological and bioavailability studies. Even greater effort isrequired to obtain multi-gram quantities of higher oligomers (hexamersthrough decamers) for similar studies since their concentration in thenatural product is much less than the pentamer. Additionally, increasingoligomeric size increases structural complexity. Factors such as thechirality of the monomeric units comprising the oligomer at differentinterflavan linkage sites, dynamic rotational isomerization of theinterflavan bonds, conformational states of the pyran ring, and themultiple points of bonding at nucleophilic centers pose efficiencyconstraints on current analytical methods of separation and purificationfor subsequent identification.

For instance, previous attempts to couple monomeric units in freephenolic form using mineral acid as the catalyst in aqueous media havemet with limited success. The yields were low, the reactions proceededwith poor selectivity, and the oligomers were difficult to isolate.(Stynberg, P. J., Nel, R. J., and Ferreira, D., Tetrahedron, 54,8153-8158 (1998); Botha, J. J., Young, D. A., Ferreira, F., and Roux, D.J. J., J. Chem. Soc., Perkins Trans. 1, 1213-1219 (1981)).

Benzylated monomers have been prepared by methods described by Kawamoto,H., Nakatsubo, F. and Murkami K., Mokuzai Gakkashi, 37, 741-747 (1991)where benzyl bromide was used in combination with potassium carbonate(K₂CO₃), and dimethyl formamide (DMF). The yield, however, was onlyabout 40%. In addition, competing C-benzylation leads to a mixture ofproducts which makes isolation of the target monomer more difficult.Also, partial racemization of (+)-catechin at both the C-2 and C-3positions was observed (Pierre, M.-C. et al., Tetrahedron Letters, 38:32, 5639-5642 (1997)).

Two primary methods for oxidative functionalization are taught in theliterature (Betts, M. J., Brown, B. R. and Shaw, M. R., J. Chem. Soc.,C. 1178 (1969); Steenkamp, J. A., Ferreira, D. and Roux, D. J.,Tetrahedron Lett., 26, 3045-3048 (1985)). In the older method, protected(+)-catechin was treated with lead tetraacetate (LTA) in benzene toproduce the 4β-acetoxy derivative which was then successfully hydrolyzedto the 3,4-diol. Flavan-3,4-diols are incipient electrophiles in thebiomimmetic synthesis of procyanidins. However, flavan 3,4-diols whichhave an oxygen functionality at the C-5 position are not available fromnatural sources and have to be synthesized. Oxidative functionalizationof the prochiral benzylic position to form the 3,4-diols thus offersconsiderable potential in the synthesis of procyanidins. The majordrawback of this reaction was a low yield (30-36%) of the acetate duringthe LTA oxidation. The more recent method of oxidatively functionalizingthe C-4 position relies on the use of2,3-dichloro-5,6-dicyano-1,4-benzoquinone (DDQ). In this method, theprotected monomer was treated with DDQ in methanol. This allowsintroduction of a methoxy group at the C4 position in a stereospecificmanner. The yield is about 40-50%.

There are a number of reports on the coupling reaction between monomersand their 3,4-diols in aqueous acid. These methods are unsatisfactorybecause of low yields, lack of specificity, and difficulty in thepurification from aqueous media. Kawamoto, H., Nakatsubo, F. andMurakami, K., J. of Wood Chem. Tech., 9, 35-52 (1989) report thetitanium tetrachloride (TiCl₄) mediated coupling between 4-hydroxyltetra-O-benzyl (+)-catechin and 5 equivalents (eq) of tetra-O-benzyl(+)-catechin to produce a 3:2 mixture of 4α→8 and 4β→8 dimers.

Hence, there is a need for synthesis methods which provide largequantities of structurally defined oligomers for in vitro and in vivoassessment. Such synthesis methods can lead to the creation of multipleconfigurational oligomers, some identical to those found in nature, aswell as rare or “unnatural” types. Accordingly, it would be advantageousto develop a versatile synthetic process capable of providing largequantities of any desired procyanidin oligomer.

SUMMARY OF THE INVENTION

A process for the preparation of a partially protected procyanidin dimeris provided. It comprises the steps of:

-   -   (a) protecting each phenolic hydroxyl group of a catechin        monomer or an epicatechin monomer with a removable protecting        group which does not deactivate the A ring of the monomer,        wherein the protecting step is carried out in an aprotic        solvent;    -   (b) optionally blocking the C-8 position of the monomer of        step (a) with a halo group;    -   (c) activating the monomer of step (a) or step (b) by        introducing an acyloxy group at the C-4 position using a        lead (IV) salt of an organic acid; and    -   (d) catalytically coupling the monomer of step (c) with an        unprotected catechin monomer or an unprotected epicatechin        monomer to form the dimer.    -   (c) coupling the activated compound of step (b) with an        unprotected procyanidin monomer in the presence of a coupling        catalyst to produce the dimer.

A process is also provided for the preparation of a linear procyanidinoligomer having 4→8 linkages. It comprises the steps of:

-   -   (a) preparing a partially protected (4→8) procyanidin dimer,        where the phenolic hydroxyl groups of the top mer are protected        with a removable protecting group which does not deactivate the        A ring of the protected mer;    -   (b) masking the the dimer of step (a) to form a dimer where the        phenolic hydroxyl groups of the top mer are protected, where the        phenolic hydroxyl groups of the bottom mer are masked, and where        the hydroxyl groups at the C-3 positions of both mers are masked        with a removable masking group which deactivates the bottom mer;    -   (c) deprotecting the dimer of step (b) to form a deprotected,        masked dimer where the phenolic hydroxyl groups of the top mer        are unprotected, where the phenolic hydroxyl groups of the        bottom mer are masked, and where the hydroxyl groups at the C-3        positions of both mers are masked;    -   () catalytically coupling the dimer of step (c) with a protected        catechin monomer or a protected epicatechin monomer having an        acyloxy activating group at the C-4 position to form a (4→8)        trimer where the phenolic hydroxyl groups of the top mer are        protected, where the phenolic hydroxyl groups of the middle mer        are unprotected, where the phenolic hydroxyl groups of the        bottom mer are masked, and where the phenolic hydroxyl groups at        the C-3 positions of the middle and bottom mers are masked;    -   (e) masking the trimer of step (d) to form a trimer where the        phenolic hydroxyl groups of the top mer are protected, where the        phenolic hydroxyl groups of the middle mer and bottom mers are        masked, and where the hydroxyl groups at the C-3 positions of        all the mers are masked;    -   (f) deprotecting the trimer of step (e) to form a trimer where        the phenolic hydroxyl groups of the top mer are unprotected,        where the phenolic hydroxyl groups of the middle and bottom mers        are masked, and where the hydroxyl groups at the C-3 positions        of all the mers are masked;    -   () catalytically coupling the trimer of step (f) with a        protected catechin monomer or a protected epicatechin monomer        having an acyloxy activating group at the C-4 position to form a        (4→8) tetramer; and    -   (h) optionally repeating the masking, deprotecting, and coupling        steps to form a higher oligomer where the number of mers are 5to        18.

In the following illustrative compounds, P is a protecting group, B is ablocking group, and M is a masking group. The following compound isillustrative of a partially protected procyanidin dimer such as thatformed in step (a) above.

The following compound is illustrative of a protected masked dimer suchas that formed in step (b) above.

The following compound is illustrative of a deprotected masked dimersuch as that formed in step (c) above.

The following compounds are illustrative of a protected, masked, blockedlinear trimer and a protected, masked, blocked linear trimer.

The following compounds are illustrative of the unblocked and blockedprotected, masked linear trimers of step (d).

The following compounds are illustrative of the unblocked and/orblocked, deprotected, masked linear trimers of step (e).

The following compounds are illustrative of compounds which result fromrepeating or alternating steps (a) to (f) to prepare higher oligomerswherein the number of mers is 4.

A process is also provided for the preparation of branched procyanidinoligomers. It comprises the steps of:

-   -   (a) preparing an unblocked or blocked, partially protected        procyanidin dimer, wherein the phenolic hydroxyl groups of the        top mer are protected with a removable protecting group which        does not deactivate the A ring of the protected mer while the        bottom mer has free phenolic hydroxyl groups;    -   (b) coupling the dimer of step (a) with a unblocked or blocked,        protected, activated procyanidin monomer to form a branched        trimer;    -   (c) deprotecting the trimer of step (b); and    -   (d) optionally carrying out one of the following steps in a        sequential, alternating, or combinational fashion to provide        procyanidin oligomers having 4 to 18 mers comprising (4→8),        (4→6) (6→4), and/or (8→4) linkages;        -   i. coupling the oligomer of step (c) with an unblocked or            blocked, protected procyanidin monomer;        -   ii. masking the oligomer of step (c), deprotecting the            masked oligomer, and coupling the masked oligomer with an            unprotected or protected, blocked activated procyanidin            monomer.

The free phenolic forms of the procyanidin dimers, linear procyanidinoligomers, or branched procyanidin oligomers are obtained bydeprotecting the dimers or oligomers and, if necessary, demasking and/ordeblocking the dimers or the oligomers. The dimers or oligomers maycontain the same or different epicatechin or catechin mers. Preferably nis 5-12, more preferably n is 5. In the linear oligomers the linkagesare (4→6) or (6→4) or are (4→8) or (8→4). In the branched oligomers atleast one of the linkages is (4→6) or (6→4)and at least one of thelinkages is (4→8).

The protecting groups may be benzyl, p-methoxybenzyl, t-butyl, ortrityl; benzyl is preferred. An aprotic solvent, e.g.,dimethylformamide, dimethylacetamide, or dimethyl sulfoxide, is used inthe protecting step; dimethylacetamide is preferred. The acyloxyactivating groups are typically acetoxy, formyloxy or propionloxy;acetoxy is preferred. The activation is carried out using a lead (IV)salt, e.g, lead tetraacetate, lead tetraformate, or leadtetraproprionate. Preferably, the activating step is carried out alsousing an organic acid which is the same as that used in the preparationof the lead salt. Suitable organic acids include formic acid, aceticacid, and propionic acid. The preferred solvent for the activating stepis benzene. The blocking group is a halo group, preferably a bromo or aniodo group. The deblocking step is carried out with an alkyl lithium,e.g., tert-butyl lithium or n-butyl lithium. The demasking step iscarried out by base hydrolysis. The deprotecting step is carried out byhydrogenolysis.

A doubly branched oligomer having the structure:

can be prepared by a process which comprises the step of:

-   -   (a) protecting each phenolic hydroxyl group of a first        procyanidin monomer with a first removable protecting group        which does not deactivate the A ring of the monomer, wherein the        protecting step is carried out in an aprotic solvent to provide        a protected monomer;    -   (b) activating for coupling the C4 position of the compound of        step (a) by introducing an acyloxy group using a lead salt of an        organic acid to provide an activated, protected monomer;    -   (c) coupling the compound of step (b) with an unprotected        procyanidin monomer in the presence of a coupling catalyst to        provide a partially protected dimer;    -   (d) masking the dimer of step (c) to provide a masked, partially        protected dimer;    -   (e) deprotecting the masked, partially protected dimer of        step (d) to provide a masked dimer;    -   (f) coupling the masked dimer of step (e) with a 4β-acetoxy        protected procyanidin monomer to produce a trimer;    -   (g) coupling the trimer of step (f) with a 4β-acetoxy        procyanidin monomer to produce a procyanidin tetramer;    -   (h) demasking the tetramer of step (g); and    -   (i) coupling the tetramer of step (h) with a 4β-acetoxy        procyanidin monomer to produce a procyanidin pentamer.

Steps to (i) can be repeated to produce a multiply branched procyanidinoligomer comprising n mers, where n is an integer from 6 to 18.

A process for producing a procyanidin dimer having a (8

8) linkage is provided. It comprises the steps of:

-   -   (a) reacting a first 8-bromo protected monomer with a hexaalkyl        distannane in the presence of palladium_((o)) to provide a        protected monomer-8-trialkyl stannane;    -   (b) coupling the compound of step (a) with a second 8-bromo        protected monomer with tetrakis (triphenyl phosphine)        palladium_((o)) in benzene to produce a (8        8) coupled dimer; and    -   (c) deprotecting the compound of step (b) to produce the (8        8) dimer.

A process is also provided for producing a procyanidin dimer having a (6

6) linkage. The process comprises the steps:

-   -   (a) reacting a first 6-bromo protected monomer with a hexaalkyl        distannane in the presence of palladium_((o)) tin to provide a        protected monomer-6-trialkyl stannane;    -   (b) coupling the compound of step (a) with a second 6-bromo        protected monomer with tetrakis (triphenyl phosphine)        palladium_((o)) in benzene to produce a (6        6) coupled dimer; and    -   (c) deprotecting the compound of step (b) to produce the (6        6) dimer.

A process is also provided for producing a procyanidin dimer having a (6

8) linkage. The process comprises the steps of:

-   -   (a) reacting a first 6-bromo protected monomer with hexaalkyl        distannane in the presence of palladium_((o)) to provide a        protected monomer-6-trialkyl stannane;    -   (b) coupling the compound of step (a) with a second 8-bromo        protected monomer with tetrakis (triphenyl phosphine)        palladium_((o)) in benzene to produce a (6        8) coupled dimer; and    -   (c) deprotecting the compound of step (b) to produce the (6        8) dimer.

A process is also provided for producing a procyanidin dimer having a (8

6) linkage. The process comprises the steps:

-   -   (a) reacting a first 8-bromo protected monomer with a hexaalkyl        distannane in the presence of palladium_((o)) to provide a        protected monomer-8-trialkyl stannane;    -   (b) coupling the compound of step (a) with a second 6-bromo        protected monomer with tetrakis (triphenyl phosphine)        palladium_((o)) in benzene to produce a (8        6) coupled dimer; and    -   (c) deprotecting the compound of step (b) to produce the (8        6) dimer.

The present processes offer important advantages and efficiencies overearlier processes for preparing procyanidin oligomers, these includebetter yields, better selectivity, and easier product isolation. Bycarrying out the protecting step in dimethylacetamide instead ofdimethyl formamide, the partial and full protection of the phenolichydroxyl groups is more readily controlled.

The present invention further provides crystalline8-bromo-tetra-O-benzyl (−)-epicatechin when dimethylacetamide is used asthe solvent in the protecting step.

DETAILED DESCRIPTION OF THE INVENTION

Monomers comprising procyanidins have the structure:

Procyanidins include those found in cocoa beans obtained from Theobromacacao and various related cocoa species, as well as the genus Herraniaand their inter- and intra-genetic crosses.

Monomers comprising procyanidins include (+)-catechin, (−)-epicatechinand their respective epimers (e.g. (−)-catechin and (+)-epicatechin).

Synthetic linear and/or branched oligomers having the followingstructures are illustrative of those that can be prepared by the aboveprocess.

In the following linear oligomers n is an integer from 0 to 16.

In the following branched oligomers, the mers A and B can be from 1 to15, with the total number of mers in the final oligomer being from 3 to18.

In the oligomers n is an integer from 2 through 18, preferably 3 through12, more preferably 5 through 12, and most preferably 5. The oligomershave interflavan linkages of(4→6)and and/or (4→8). The oligomersprepared by the inventive process may be represented by the structuresabove. For the linear oligomer, when x is 0, the oligomer is termed a“dimer”; when x is 1 the oligomer is termed a “trimer”; when x is 2, theoligomer is termed a “tetremer”; when x is 3, the oligomer is termed a“pentamer”; and similar recitations may be designated for oligomershaving x up to and including 16 and higher, such that when x is 16, theoligomer is termed an “octadecamer.” For the branched oligomer, when B Aor B is 1, the oligomer is termed a “trimer”; with similar recitationssuch as those described for the linear oligomers.

Stereoisomers of the oligomers are encompassed within the scope of theinvention. The stereochemistry of the monomers comprising an oligomercan be described in terms of their relative stereochemistry, i.e.,“alpha/beta” or “cis/trans”, or in terms of their absolutestereochemistry, i.e., “R/S”. The term “alpha” (α) indicates thesubstituent is oriented below the plane of the flavan ring, whereas theterm “beta” (β) indicates that the substituent is oriented above theplane of the ring. The term “cis” indicates two substituents areoriented on the same face of the ring, whereas the term “trans”indicates two substituents are oriented on opposite faces of the ring.The terms “R” and “S” are used to denote the arrangement of thesubstituents about a stereogenic center, based on the ranking of thegroups according to the atomic number of the atoms directly attached tothe stereogenic center (CIP convention).

There are multiple stereochemical linkages between position C-4 of aflavan 3-ol monomer and positions C-6 and C-8 of the adjacent monomer.The stereochemical linkages between monomeric units is designated hereinas (4α→6) or (4β→6) or (4α→8) or (4β→8) for linear oligomers. Forlinkages to a branched or junction monomer, the stereochemical linkagesare (6→4α) or (6→4β) or (8→4β) or (8→4β). When (+)-catechin, designatedherein as C, is linked to another C or to (-)-epicatechin, designatedherein as EC, the linkages are advantageously (4α→6) or (4α→8). When ECis linked to C or another EC, the linkages are advantageously (4β→6) or(4β→8).

Linear and branched oligomers can be prepared by the methods of thepresent invention using the steps of protecting, activating, coupling,masking, blocking, deprotecting, demasking and deblocking. In eachreaction sequence the catechin or epicatechin monomers can be used toprepare linear or branched oligomers containing the same or differentmonomers. Higher oligomers can be prepared by repeating the coupling ofa dimer, trimer, etc. with an additional catechin or epicatechin monomerusing the above steps.

Examples of the compounds which can be synthesized according to themethod of the invention include dimers;

EC-(4β→8)-EC and EC-(4β→6)-EC, wherein EC-(4β→8)-EC is preferred;trimers [EC-(4β→8)]₂-EC, [EC-(4β→8)]₂-C, and [EC-(4β→6)]₂-EC, wherein[EC-(4β→8)]₂-EC is preferred; tetramers [EC-(4β→8)]₃-EC, [EC-(4β→8)]₃-C,and [EC-(4β→8)]₂-EC-(4β→6)-C wherein [EC-(4β→8)]₃-EC is preferred; andpentamers [EC-(4β→8)]₄-EC, [EC-(4β→8)]₃-EC-(4β→6)-EC,[EC-(4β→8)]₃-EC-(4β→8)-C, and [EC-(4β→8)]₃-EC-(4β→6)-C, wherein[EC-(4β→8)]₄-EC is preferred. An example of a branched trimer is

an example of a branched tetramer is

an example of a branched pentamer is

Additional compounds which can be synthesized include the following:

(i) a hexamer, wherein one monomer (C or EC) is linked to a pentamercompound listed above, e.g., [EC-(4β→8)]₅-EC, [EC-(4β→8)]₄-EC-(4β→6)-EC,[EC-(4β→8)]₄-EC-(4β→6)-C, and [EC-(4β→8)]₄-EC-(4β→6)-C; wherein[EC-(4β→8)]₅-EC- is preferred, with an example of a branched hexamerbeing

(ii) a heptamer, wherein any combination of two monomers (C and/or EC)is linked to one of the above pentamers, e.g., [EC-(4β→8)]₆-EC,[EC-(4β→8)]₅-EC-(4β→6)-EC, [EC-(4β→8)]₅-EC-(4β→8)-C, and[EC-(4β→8)]₅-EC-(4β→6)-C, wherein [EC-(4β→8)]₆-EC is preferred with anexample of a branched heptamer being

(iii) an octamer, wherein any combination of three monomers (C and/orEC) is linked to one of the above pentamers, e.g., [EC-(4β→8)]₇-EC,[EC-(4β→8)]₆-EC-(4β→6)-EC, [EC-(4β→8)]₆-EC-(4β→6)-C, wherein[EC-(4β→8)]₇-EC is preferred with an example of a branched octamer being

(iv) a nonamer, wherein any combination of four monomers (C and/or EC)is linked to one of the above pentamers, e.g., [EC-(4β→8)]₈-EC,[EC-(4β→8)]₇-EC-(4β→6)-EC,[EC-(4β→8)]₇-EC-(4β→8)-C,[EC-(4β→8)]₇-EC-(4β→6)-C, wherein[EC-(4β→8)]₈-EC is preferred with an example of a branched nonamer being

(v) a decamer, wherein any combination of five monomers (C and/or EC) islinked to one of the above pentamers, e.g., [EC-(4β→8)]₉-EC,[EC-(4β→8)]₈-EC-(4β→6)-EC, [EC-(4β→8)]₈-EC-(4β→8)-C,[EC-(4β→8)]₈-EC-(4β→6)-C, wherein [EC-(4β→8)]₉-EC is preferred with anexample of a branched decamer being

(vi) an undecamer, wherein any combination of six monomers (C and/or EC)is linked to one of the above pentamers, e.g., [EC-(4β→8)]₁₀-EC,[EC-(4β→8)]₉-EC-(4β→6)-EC, [EC-(4β→8)]₉-EC-(4β→8)-C,[EC-(4β→8)]₉-EC-(4β→6)-C, wherein [EC-(4β→8)]₁₀-EC is preferred with anexample of a branched undecamer being

(vii) a dodecamer, wherein any combination of seven monomers (C and/orEC) is linked to one of the above pentamers, e.g., [EC-(4β→8)]₁₁-EC,[EC-(4β→8)]₁₀-EC-(4β→6)-EC, [EC-(4β→8)]₁₀-EC-(4β→8)-C,[EC-(4β→8]₁₀-EC-(4β→6)-C, wherein [EC-(4β→8)]₁₁-EC is preferred with anexample of a branched dodecamer being

The aforementioned list of oligomers is illustrative and is provided toillustrate the types of compounds that can be prepared by the methods ofthe invention and is not an exhaustive list of compounds encompassed bythe invention. The oligomers can be separated and purified by themethods disclosed in U.S. Pat. No. 5,554,645 issued Sep. 10, 1976 toRomanczyk et al. and U.S. Pat. No. 5,712,305 issued Jan. 27, 1998 toRomanczyk et al.

One skilled in the art will appreciate the rotation of a number of bondswithin an oligomer can be restricted due to steric hindrance,particularly if the oligomer is substituted, such as with benzyl groups.Accordingly, all possible regioisomers and stereoisomers of thecompounds prepared by the invention are encompassed within the scope ofthe invention.

Definitions

As used herein, a “protecting group” is a removable group which replacesthe hydrogen of the phenolic hydroxyl groups in the procyanidin monomersor oligomers. The group should be removable under conditions which donot affect the procyanidin oligomers.

As used herein, a “blocking group” is a removable group which directsthe coupling by blocking the C-8-position of the A ring of a catechin orepicatechin monomer or a procyanidin oligomer, thus directing couplingwith another procyanidin monomer to occur at the C-6 position of the Aring. The group should be removable under conditions that do not affectthe procyanidin oligomer.

As used herein, a “masking group” is a removable group which masks theunprotected phenolic hydroxyl and the C-3 hydroxyl group(s) of aprocyanidin monomer or higher oligomer during the coupling of the dimeror higher oligomer with another procyanidin monomer. The group should beremovable under conditions that do not affect the procyanidin oligomer.

As used herein, an “activating group” is an acyloxy group whichactivates the C-4position of the C ring of a procyanidin monomer, dimer,or higher oligomer and results in coupling with another procyanidinmonomer or oligomer at that position.

The term “combinational” used herein refers to the possibleregiochemical linkage possibilities for preparing any given procyanidinoligomer. For instance, a linear procyanidin tetramer can be comprisedof (4→8) and (4→6) linkages between the monomers comprising thetetramer. For synthesis purposes these linkages result in separatecompounds which can have different biological activity. Forstructure-activity studies it would be advantageous to provide a seriesof these oligomers to determine the importance of regiochemical linkagesto biological activity. For a linear tetramer the possible linkages areas follows:

Tetramer 1 Tetramer 2 Tetramer 3 Tetramer 4 Tetramer 5 Tetramer 6Tetramer 7 Tetramer 8 4 → 8 4 → 6 4 → 8 4 → 8 4 → 6 4 → 6 4 → 6 4 → 8 4→ 8 4 → 6 4 → 8 4 → 6 4 → 8 4 → 6 4 → 8 4 → 6 4 → 8 4 → 6 4 → 6 4 → 8 4→ 8 4 → 8 4 → 6 4 → 6which necessitate the need for a synthetic procedure to prepare 8different tetramers, each requiring different steps for preparation.

Protecting Groups

The protecting groups useful in this invention are electron donatingmoieties that function to activate catechin and epicatechin monomers atthe C-4 position. In the C-4 activation reaction, electron donatingphenolic protecting groups function to stabilize, and thereby assist inthe formation of the intermediate C-4 benzylic cation formed byoxidation of the protected monomer with a lead (IV) salt. In thecoupling reaction, an electrophilic aromatic substitution reaction, theelectron donating phenolic protecting groups function again tostabilize, and thereby assist in the formation of the C-4 benzyliccation by treatment of the C-4 acyloxy substituted catechin orepicatechin monomer (an activated monomer) with a Lewis acid catalyst.In the coupling reaction, the electron donating phenolic protectinggroups also function to increase the differences in reactivity betweenthe various aryl moieties that may be present in the reaction. Asdescribed below, unprotected catechin or epicatechin monomers orselected unprotected (deprotected) monomeric units of a procyanidinoligomer are used as nucleophiles in the coupling reaction. TheC-4acyloxy substituted, protected catechin or epicatechin monomer, ontreatment with a Lewis acid, functions as the electrophile. Theunprotected procyanidins function as nucleophiles because they processhigher election densities, that is higher nucleophilicities, than theprotected procyanidin monomers. Any self-coupling between protectedprocyanidin monomers is limited due to the comparatively highernucleophilicities of the unprotected procyanidins.

Among the various protecting groups, benzyl groups are preferred becausethey are more easily removed under mild conditions such ashydrogenolysis. Another advantage of benzylation (except, for example,p-nitro benzylation) is that it will not deactivate the aromatic ringtoward coupling when the procyanidin monomers or oligomers are acting asnucleophiles. Surprisingly and quite unexpectedly, changing the aproticorganic solvent used in the protecting step from dimethyl formamide(DMF) to dimethyl acetamide (DMA) resulted in an increased yield of theprotected oligomer, perhaps due to the fact that the slightly higherdielectric constant of DMA may be favoring the O-alkylation. The yieldwas at least about 50%, typically about 60% to about 70%. In addition,no extra clean-up procedures were required and the products were readilycrystallized. Examples 1 and 2 describe the specific conditions for thepreparation of tetra-O-benzyl-(+)-catechin andtetra-O-benzyl-(−)-epicatechin. Further investigation of the solventsystem indicated that potassium carbonate (K₂CO₃) was preferred oversodium carbonate (Na₂CO₃) because of its solubility within the preferredsolvent system. It was found that potassium iodide can be used incatalytic amounts in combination with benzyl bromide.

Another useful protecting group for (−)-epicatechin is p-methoxy benzyl(PMB) groups. If PMB is selected as a protecting group in preparing apartially protected procyanidin dimer, then the protecting step furthercomprises acetylating the procyanidin monomer followed by treatment withsodium hydride, PMB-chloride and DMF in water to remove the phenolicacetate groups, resulting in alkylation of the phenoxide ions with PMB.When utilizing DMA as the solvent in the protecting step, PMB groupsshould not be used. Tetra-O-PMB-(−) epicatechin can be prepared usingthe procedure by Kawamoto, H., Nakatsubo, F. and Murakami, K. Synth.Commun., 26, 531-534 (1996). Pentaacetyl(−)-epicatechin is firstprepared (as described in Example 3 below) by treatment with sodiumhydride (NaH), p-methoxybenzyl chloride (PMBC1), dimethyl formamide(DMF) and water in an amount sufficient (4 eq) to generate theequivalent amount of base to remove the four phenolic acetate groups insuccession. The resultant phenoxide ions undergo rapid alkylation withthe PMBC1. Examples 4 and 5 describe the specific conditions by whichpenataacetyl (+)-catechin and tetra-O-p-methoxybenzyl-3-acetyl-(−)epicatechin were prepared.

The skilled artisan will recognize that other protecting groups such astert-butyl, trityl and 2,4-dimethoxy benzyl can be used

C-4 Activation

Altering the LTA reaction conditions to 1:1 benzene:acetic acideventually produced the highest yield (60-70%) and the stereospecific 4βproduct. Other useful solvent mixtures include benzene, toluene,chlorobenzene, cyclohexane, heptane, carbon tetrachloride, or mixturesthereof, admixed with an organic acid, which is the same as that used toprepare the lead (IV) salt used in the activation reaction.

The lead salts of organic acids are employed in the activation step,e.g., lead tetraformate, lead tetrapropionate, and the like. Preferably,the corresponding organic acids, i.e., formic and propionic acids, areused in combination with the lead salt to improve the yield. Thepreferred salt is lead acetate and the preferred combination is leadtetraacetate and acetic acid.

Examples 6-9 describe the specific conditions for the preparation of4β-acetoxy tetra-O-benzyl-(−) -epicatechin, 4β-hydroxyltetra-O-benzyl-(+)-catechin, 4βhydroxyl tetra-O-benzyl-(−)-epicatechin,and 4β-acetoxy tetra-O-benzyl-(+)-catechin.

Masking Groups

The masking groups useful in this invention are electron withdrawingmoieties that function to deactivate selected monomeric units ofprocyanidin oligomers in the electorphilic aromatic substitutioncoupling reaction described hereinbelow. When procyanidin oligomers areused in the coupling reaction, it is imperative that an activatedmonomer does not randomly react with different monomeric units of theoligomer. Masking groups are used to increase the differences inreactivity between the different monomeric units of an oligomer. Use ofelectron withdrawing moieties as masking groups deactivates themonomeric units of the oligomer bearing the masking groups towardscoupling with an activated, protected monomer. Accordingly, in theprocess of this invention, an activated monomer selectively reacts withan unprotected monomeric unit of a partially masked oligomer due to thehigh reactivity (nucleophilicity) of the unprotected monomeric unit andthe reduced reactivity of the masked monomeric units of the oligomer.

Masking groups that are useful in the process of this invention includeacyl groups such as acetyl and propionyl, aroyl groups such as benzoyl,carbamate groups such as N-phenyl carbamate, carbonate groups such asmethyl carbonate, and arylsulfonyl groups such as2,4-dinitrobenzenesulfonyl. Preferably, the masking group is acetyl.Masking of unprotected phenolic hydroxyl and C-3 hydroxyl groups ofprocyanidin oligomers may be accomplished using any conventionaltechnique for replacing the hydrogen of the hydroxyls with suitablemasking groups such as those identified above. The reagents used willdepend on the masking groups being introduced and are well known in theart.

Coupling Reaction

In the process of this invention, the use of a coupling catalyst such asa Lewis acid (e.g. lithium bromide) is preferred. The use of a4β-acetoxy derivative as the electrophile is also preferred. Theselectivity of the coupling reaction is significantly improved thereby.The use of the Li⁺as a counter ion favored C-alkylation overO-alkylation.

When methanol is added to a refluxing methylene chloride solution of4βacetoxytetra-O-benzyl-(+)-catechin and LiBr, 4β-methoxytetra-O-benzyl-(+)-catechin is formed in significantly higher yield (seeExample 10). The β stereochemistry is assigned on the basis of thecoupling constant of 3.5 Hz between H-3 and H-4 which indicates a cisrelationship. This reaction does not occur when a halide such as LiBr isnot employed as the Lewis acid. In this reaction, the acetoxy isdisplaced by the halide which then immediately reacts with the methanol,acting as a nucleophile, thereby driving the reaction to equilibrium.

It was unexpected that the use of LiBr as the Lewis acid would drive thecoupling reaction between 4β-acetoxy tetra-O-benzyl monomer and anothermonomer acting as the nucleophile, thereby eliminating the step ofpreparing a 3,4 diol of the monomer.

To test this unexpected finding and gain insight into the potentialapplication of this reaction, 4β-acetoxy tetra-O-benzyl-(+)-catechin wasreacted with (−)-epicatechin in the presence of LiBr as described inExample 11. The resultant dimer obtained was 90% pure and the yield was62%. The ¹H NMR spectrum indicated one singlet at δ 5.85 for 1H and apair of doublets at δ 6.19 and 6.16 each integrating to 1H with atypical m-coupling of 1.5 Hz which indicated the formation of only oneisomer. Treatment of the dimer with acetic anhydride/pyridine formed thehexaacetate (Example 13) and, as expected, the singlet for the C-6′hydrogen shifted downfield to δ 6.52. The doublet for the C-4 hydrogenhad a coupling constant of 9.6 Hz, indicating the a configuration. Thepartially benzylated dimer was deprotected with hydrogen/palladium_((o))(H₂/Pd) to obtain the dimer (+)-catechin-(4α→8)-(−)-epicatechin (seeExample 12). HPLC analysis indicated that, in addition to the abovedimer, another unknown dimer (13.5%) was present as well as a traceamount of trimer and tetramer. Final confirmation of the dimer'sstructure was made by preparing the octaacetate and comparing the ¹H NMRto the literature (Kawamoto, H., Nakatsubo, F. and Murakami, K., J. WoodChem. Tech., 9, 35-92 (1989)).

To study the surprising results using the LiBr coupling reaction, thecoupling between 4β-acetoxy tetra-O-benzyl-(−)-epicatechin and(−)-epicatechin was performed as described in Example 15 (refer toExample 17 for the catechin dimer). The dimer(−)-epicatechin-(4β→8)-(−)-epicatechin was obtained after hydrogenolysis(Example 16). As shown in the following table, appreciable amounts oftrimers and tetramers occurred in this reaction.

TABLE 1 LiBr Coupling Reaction Monomer 1 Monomer 2 % Dimer* % Trimer* %Tetramer* 4β-acetoxy tetra-O-benzyl (−)-epicatechin 71.4, 13.5 3.8 4.7(+)-catechin 4β-acetoxy tetra-O-benzyl (−)-epicatechin 44.7 16.8, 9.34.5, 3.3 (−)-epicatechin 4β-acetoxy tetra-O-benzyl (+)-catechin 68.0** —— (+)-catechin *HPLC-MS analysis in negative ion mode (NH₄OH) @ 0.04mL/min; fragmentor 75; Vcap 3000. (See also FIGS. 1A and 1B). **Yieldbased on silica gel chromatography.This table indicates that only one primary dimer results from thisreaction. The manipulation of reaction time and amounts of reactantmonomers can reduce the presence of higher oligomers. Additionally, ithas been observed that tetra benzyl-monomers do not act as nucleophilesin the lithium bromide coupling reaction. The free phenolic hydroxylgroups are necessary to increase the activity of the aromatic ringtowards coupling. This is significant, since it offers a means todifferentiate between rings capable of participating in the coupling andrings not capable of participating in the coupling.

Coupling reaction yields have also been improved by using lithium iodide(Li I) as Lewis Acid (see Example 20). Also, the reaction between4β-acetoxy tetra-O-benzyl-(+)-catechin and (−)-epicatechin was completedin only 18 hours with a yield of 85% after chromatography. In Example 11where lithium bromide was used, and the yield was only 62% after 24hours.

This coupling procedure can be used for higher oligomers other than thedimer as shown in the following reaction schemes.

Since the phenolic hydroxyl groups in the A ring are benzylated,coupling occurs only at the C-6′ position of the D ring and leads to theformation of a branched trimer. This was successfully tested by reactingtetra-O-benzyl-(+)-catechin-(4β→8)-(−)-epicatechin (see Example 11) with4β-acetoxy tetra-O-benzyl -(+)-catechin (see Example 6) with LiBr inTHF-methylene chloride. A spot was isolated from Thin LayerChromatography (TLC) where the ¹H NMR was too complicated to interpret.However, mass spectral analysis showed that the molecular ion peak atm/z 1861 corresponded to the desired structure in which one of thehydroxyl groups was not acetylated. The reaction was repeated and thesame product isolated. The mass spectrum clearly showed the formation ofthis branched trimer where (M+Na)⁺ at m/z 1610 and (M+H)⁺ at m/z 1588were observed with typical retro Diels Alder cleavage fragmentation. Athird replicate reaction was conducted for a longer time and again themass spectrum was consistent with a branched trimer whose tentativestructure was assigned astetra-O-benzyl-(+)-catechin-(4α→8)-(−)-epicatechin-(6→4α)-tetra-O-benzyl-(+)-catechin(see Example 24).

For the synthesis of linear oligomers, a strategy of selectiveactivation-deactivation of the rings capable of participating in thecoupling reaction was developed. In this case, the partially benzylateddimer was acetylated and then hydrogenolysed, allowing the preparationof a dimer with free phenolic hydroxyl groups (OH groups) in the A/Brings, and with acetate groups in the D/E rings. The electronwithdrawing acetyl groups deactivated the D ring and thus allowedcoupling with the 4β-acetoxy monomers to occur regioselectively at theC-8 position of the A ring. The resultant trimer can be subjected to thesame process of acetylation and debenzylation followed by coupling withanother 4β-acetoxy monomer to produce a tetramer. Repetitions of thesesteps leads to oligomers of increasing size.

This process was confirmed where the partially protected dimer ofExample 11 was acetylated (see Example 13) and then hydrogenolysed (seeExample 14). Doubling of NMR peaks was observed which is indicative ofrotamers. NMR spectra taken at higher temperatures (313° K) simplifiedthe spectrum, confirming the existence of the rotamers. Interestingly,performing the hydrogenolysis in ethyl acetate enabled the isolation ofmono-O-benzyl-3-acetyl-(+)-catechin (4α→8)-pentaacetyl-(−)-epicatechin.When this oligomer was hydrogenolysed, the product was the same as thatobtained previously. Reacting3-acetyl-(+)-catechin-(4α→8)-pentaacetyl-(−) -epicatechin (see Example14) with 4β-acetoxy tetra-O-benzyl-(−)-catechin (see Example 6) resultedin the desired product, i.e., tetra-O-benzyl (+)-catechin(4α→8)-3-acetyl (+)-catechin (4α→8)-pentaacetyl(−) -epicatechin (seeExample 20). The mass spectrum (APCI, negative ion mode) indicated astrong molecular ion peak at m/z 1479.6 which was identical to thecalculated mass for C₈₅H₇₄O₂₄ (1479.5). Mass fragments at m/z 1437, 1389and 1347 were consistent for the loss of acetyl, benzyl andacetyl/benzyl groups from the parent compound. FAB MS showed a molecularion peak at m/z 1482 (M+H)⁺and a fragmentation pattern consistent fortetra-O-benzyl-3-acetyl(+)-catechin-(4α→8) -pentaacetyl-(−)-epicatechin.

Similarly, the linear tetramer was formed by preparingtetra-O-benzyl-(+)-catechin-(4α→8)-3-acetyl-(+)-catechin-(4α→8)-pentaacetyl(−)-epicatechin(see Example 20) which was acetylated totetra-O-benzyl-3-acetyl-(+)-catechin-(4α→8)-pentaacetyl-(+)-catechin-(4α→8)-pentaacetyl(−)-epicatechin (see Example 21). Hydrogenolysis of the above compound(see Example 22) produced3-acetyl-(+)-catechin-(4α→8)-pentaacetyl-(+)-catechin-(4α→8)-pentaacetyl(−)-epicatechin. Coupling3-acetyl-(+)-catechin-(4α→8)-pentaacetyl-(+)-catechin-(4α→8)-pentaacetyl(−)-epicatechin with 4βacetoxy tetra-O-benzyl-(+)-catechin (see Example6) resulted in the formation of the tetramertetra-O-benzyl-(+)-catechin-(4α→8)-3-acetylpentaacetyl-(+)-catechin-(4α→8)-pentaacetyl-(+)-catechin-(4α→8)-pentaacetyl(−)-epicatechin (see Example 23). The FAB mass spectrum showed themolecular ion peak at m/z 1978 which is consistent with the structure.

An in situ replacement of the phenolic acetate groups with benzyl groupshas also been developed. The in situ replacement of the acetyl forbenzyl groups was conducted on3-acetyl-tetra-O-benzyl-(+)-catechin-(4α→8)-pentaacetyl-(−)-epicatechin(see Example 13) using the conditions specified in Example 18 to resultin the preparation of3-acetyl-tetra-O-benzyl-(+)-catechin-(4α→8)-3-acetyl-tetra-O-benzyl-(−)-epicatechin.Hydrogenolysis (see Example 19) provided the recovery oftetra-O-benzyl-(+)-catechin-(4α→8)-tetra-O-benzyl-(−)-epicatechin whichwas then hydrogenolysed to the free dimer, proving the feasibility ofthis procedure.

Blocking Groups

On the basis of the above steps, methods for the synthesis of (4→6)interflavan linkages between monomers have been developed. For example,monomers can be benzylated in high yields using the DMA solvent systempreviously described. The benzylated monomers can be brominated at theC-8 position to provide 8-bromo-tetra-O-benzyl monomers as shown inExamples 25 and the several variants shown in Examples 26, and 27, andExample 28 describes the absolute stereochemistry for8-bromo-tetra-O-(−)-epicatechin. Deprotecting these derivatives resultsin the preparation of the 8-bromo monomers. The resultant bromoderivative effectively blocks coupling at the C-8 position, thusdirecting coupling to occur at the C-6 position. Coupling of the 8-bromomonomers with 4β-acetoxy tetra-O-benzyl-monomer under the conditions ofthe lithium bromide procedure results in the formation of a (4→6) dimer.A typical reaction scheme is illustrated below.

Debromination (i.e., deblocking) is effected at low temperature (−78°C.) in solution with a suitable alkyl lithium compound such as n-butyllithium or tert-butyl lithium, followed by protonation of the resultantdimer by a weak protic acid (e.g., water). By employing the additionalsteps embodied in the invention, the (4→6) dimer can be extended tohigher oligomeric size, comprising variations of regio- andstereochemistry previously described.

Deprotection and Demasking

The reagents used in the deprotection step will depend upon the groupbeing removed. For example, when removing the benzyl protecting groups,hydrogenolysis is carried out using the conditions set forth in Examples12, 16 and 22. When the masking groups are removed, alkaline hydrolysisis carried out using the conditions set forth in Examples 5 and 18.

Alternatively, commercial lipase can be used to enzymaticallydeacetylate the protected oligomer. Removal of the protecting or maskinggroups can be accomplished using any conventional techniques providedthe techniques do not adversely affect the procyanidin oligomer.

Compounds of the Invention

Novel compounds that can be produced by the process of the inventioninclude novel multiple branched, preferably doubly branched, procyanidinoligomers represented by the following structure.

Other compounds that can be produced include procyanidin oligomerscomprised of (8

8), (6

8), and (6

6), linkages, where representative structures are shown below.

Procyanidin oligomer with (8

8) linkage

Procyanidin oligomer with (6

6) linkage

Procyanidin oligomer with (8

6) linkage

The steps outlined above under the Blocking Groups Section, can beextended to produce procyanidin oligomers comprising (6

6), (6

8), (8

8) interflavan linkages. These compounds can be obtained from 8-bromo-or 6-bromo-monomer intermediates. Coupling of these compounds witharylboronic or arylstannanes obtained from the same halogenatedintermediates by Suzuki coupling or by Stille reactions leads to thedesired oligomeric linkages (Suzuki, A., Pure Appl. Chem. 57,11749-11758 (1985), Stille, J. K., Agnew, Chem. Internal. Ed. Engl., 25,508-524 (1986)).

Uses of the Procyanidin Oligomers

The oligomers have the same uses, and can be formulated, purified andadministered in the same manner as described in U.S. Pat. No. 5,554,645issued Sep. 10, 1996 to Romanczyk et al. and U.S. Pat. No. 5,712,305issued Jan. 27, 1998 to Romanczyk et al. Such uses include the use asantineoplastic agents, anti-cancer agents, anti-tumor agents,antioxidants, DNA topoisomerase inhibitors, cyclo-oxygenase andlipoxygenase modulators, nitric oxide or nitric oxide synthasemodulators, non-steriodal antinflammatory agents, apoptosis modulators,platelet aggregation modulators, blood or in vivo modulators,antimicrobials, and inhibitors of oxidative DNA damage.

EXAMPLES

In the following examples, (+)-catechin and (−)-epicatechin areexemplary procyanidin monomers used to demonstrate the methods of theinvention and no limitation of the invention is implied. These monomersmay be obtained from commercial sources or isolated and purified fromnatural sources such as from the seeds of Theobroma cacao, relatedspecies, the genus Herrania and their inter- and intra-genetic crosses.Unless specified otherwise, the purity of compounds prepared in theExamples were ^(˜)85% or better.

Example 1 Preparation of Tetra-O-benzyl-(+)-catechin

To a solution of (+)-catechin (580 mg, 2 mmol) in DMA (15 mL), benzylbromide (960 μL, 4 eq) and K₂CO₃ (1.7 gm, 6 eq) were added and themixture stirred at r.t. under argon for 48 hours. The mixture was thenpartitioned between ethyl acetate and water (50 mL each). The organiclayer was washed with water (3×50 mL), then 50 mL saturated NaCl.Removal of the solvent gave a viscous residue from which the titlecompound was isolated by crystallization from 50 mL methylenechloride:methanol (9:1; v/v) to provide 880 mg of off-white crystals ata yield of 68%. ¹H NMR (CDCl₃) δ_(H) 7.44-7.24 (20H, m, Ar—H), 7.0 (1H,s, H-2′), 6.94 (2H, s, H-5′, H-6′), 6.25, 6.19 (2×1H, 2×d, J=2.0 Hz,H-6, H-8), 5.16 (4H, s, CH₂Ph), 5.0, 4.97 (2×2H, 2×s, CH₂Ph), 4.61 (1H,d, J=8.2 Hz, H-2), 3.98 (1H, m, H-3), 3.10 (1H, dd, J=16.5 Hz, H-4α),2.63 (1H, dd, J=8.9, 16.5 Hz, H-4β).

Example 2 Preparation of Tetra-O-benzyl-(−)-epicatechin

The title compound was prepared in an identical manner to that set forthin Example 1 except that (−)-epicatechin was used in place of(+)-catechin to provide 893 mg of off-white crystals at a yield of 69%.¹H NMR (CDCl₃) δ_(H) 7.43-7.30 (20H, m, Ar—H) 7.13 (1H, s, H-2′), 6.96(2H, s, H-5′, H-6′), 6.26 (2H, m, H-6, H-8), 5.18, 5.16 (2×2H, s,CH₂Ph), 5.01, 4.99 (2×2H, 2×s, CH₂Ph), 4.90(1H, s, H-2), 4.19 (1H, bm,H-3), 2.45 (2H, m, H-4), 1.64 (1H, d, J=3.8 Hz, OH).

Example 3 Preparation of Pentaacetyl (−)-epicatechin

500 mg (−)-epicatechin (1.6 mmole) was dissolved in 5 mL cold (0° C.)dry pyridine. 2 mL of acetic anhydride was added, the solution stirredfor 18 hours under an argon atmosphere. The solution was thenpartitioned between 50 mL ethyl acetate:50 mL 1N HCl and the organiclayer washed 3×50 mL with 1N HCl, followed by 50 mL water and 50 mLsaturated NaCl. The washed organic layer was dried over MgSO₄, filteredand dried to obtain a viscous oil which solidified upon addition of 100mL hexane to provide 720 mg of product at a yield of 90%. ¹H NMR (CDCl₃)δ_(H) 7.36 (1H, d, J=2 Hz, H-2′), 7.27 (1H, dd, J=2.0, 8.4 Hz, H-6′),7.20 (1H, d, J=8.4 Hz, H-5′), 6.67(1H, d, J=2.3Hz, H-6, or H-8), 6.56(1H, d, J=2.3 Hz, H-8 or H-6), 5.38 (1H, m, H-3), 5.11 (1H, bs, H-2),2.98 (1H, dd, J=4.4, 17.8 Hz, H-4), 2.87 (1H, dd, J=2, 17.8 Hz, H-4),2.299, 2.297, 2.295, 2.282, 1.920 (5×3H, 5×s, 5×COCH₃).

Example 4 Preparation of Pentaacetyl (+)-catechin

The title compound was prepared in an identical manner to that set forthin Example 3 except that (+)-catechin was used in place of(−)-epicatechin to provide 720 mg at a yield of 90%. ¹H NMR (CDCl₃)δ_(H) 7.16 (1H, d, J=2 Hz, H-2′), 7.26 (1H, dd, J=2.0, 8.4 Hz, H-6′),7.19 (1H, d, J=8.4 Hz, H-5 ′), 6.66 (1H, d, J=2.3 Hz, H-6, or H-8), 6.59(1H, d, J=2.3 Hz, H-8 or H-6), 5.25(1H, m, H-3), 5.15 (1H, d, J=6.1 HzH-2), 2.87 (1H, dd, J=5.1, 16.8 Hz, H-4), 2.63 (1H, dd, J=6.4, 16.8Hz,H-4), 2.290, 2.286, 2.279, 2.051, 2.006 (5×3H, 5×s, 5×COCH₃).

Example 5 Preparation ofTetra-O-p-methoxybenzyl-3-acetyl-(−)-epicatechin

To a mixture of pentaacetyl (−)-epicatechin (50 mg, 0.2 mmol),p-methoxybenzyl chloride (69 mg, 4.4 eq), 60% NaH in mineral oil (10 mg,4 eq) and DMF (5 mL), water (20 μL, 4 eq) was added at 0° C. dropwiseover a period of 5 minutes. After stirring under argon for 12 hours atr.t., the reaction mixture was diluted with ethyl acetate (30 mL) andwashed with water (50 mL) and 30 mL saturated NaCl. The organic layerwas dried over MgSO₄ and the solvent evaporated to obtain a pale yellowoil from which the title compound was isolated by silica gelchromatography as a white solid, crystallized from hexane:methylenechloride (1:1, v/v); yield (50 mg, 70%). ¹H NMR (CDCl₃) δ_(H) 7.35-7.29(9H, m, Ar—H), 6.92-6.85 (10H, m, Ar—H), 6.26 (2H, bs, H-6, H-8)), 5.38(1H, m, H-3), 5.06-6.85(8H, m, 4×CH₂), 4.92 (1H, s, H-2), 3.80 (12H,overlapping singlets, 4×OCH₃), 2.93 (2H, m, H-4), 1.85 (3H, s, OCOCH₃).

Example 6 Preparation of 4β-Acetoxy tetra-O-benzyl-(+)-catechin

Tetra-O-benzyl-(+)-catechin (300 mg, 0.46 mmole) and lead tetraacetate(304 mg, 1.5 eq) were combined in a round bottom flask and dried undervacuum for 30 min. Argon was introduced, followed by addition of benzeneand glacial acetic acid (5 mL each). The initial yellow color faded onaddition of acetic acid. The solution was stirred for 24 hours at r.t.and transferred to a separatory funnel. The mixture was washed with cold1N NaOH (4×50 mL), followed by water (50 mL) and finally with saturatedNaCl (50 mL). The organic layer was dried over Na₂SO₄ followed byremoval of solvent to produce a brownish residue from which silica gelchromatography furnished the title compound by elution with hexane:ethylacetate (7:3, v/v). The elute was evaporated to produce 210 mg, 66% ofthe title compound. ¹ H NMR (CDCl₃) δ_(H) 7.44-7.28 (20H, m, Ar—H), 7.08(1H, s, H-2′), 7.01, 6.95 (2H, ABq, J=8.3 Hz, H-5′H-6′),6.41 (1H, d,J=3.6 Hz, H-4), 6.23, 6.15 (2×1H, 2×d, J=2.1 Hz, H-6, H-8), 5.16 (4H, s,CH₂Ph), 5.05, 4.97 (2×2H, 2×s, CH₂Ph), 4.83 (1H, d, J=10.3 Hz, H-2),4.13 (1H, dd, J=3.6, 10.3 Hz, H-3), 2.23 (1H, bs, OH), 2.07 (3H, s,COCH₃).

Example 7 Preparation of 4β-Acetoxy tetra-O-benzyl-(−)-epicatechin

The title compound was prepared in an identical manner to that set forthin Example 6 except that 1.01 gm of tetra-O-benzyl-(−)-epicatechin (1.55mmol) was used in place of tetra-O-benzyl-(+)-catechin to provide 62 mg,59% product. ¹H NMR (CDCl₃) δ_(H) 7.44-7.24 (20H, m, Ar—H), 7.12 (1H, s,H-2′), 6.98, 6.95 (2H, ABq, J=8.3 Hz, H-5′, H-6′), 6.25 (2H, s, H-6,H-8), 5.16 (4H, s, CH₂Ph), 6.10, (1H, d, J=2.5 Hz, H-4), 5.17, 5.16,5.03 (4×2H, 4×s CH₂Ph), 4.97 (1H, s, H-2), 3.95(1H, m, H-3), 2.0 (3H, s,COCH₃).

Example 8 Preparation of 4β-Hydroxyl tetra-O-benzyl-(+)-catechin

To a solution of 4-acetoxy tetra-O-benzyl-(+)-catechin (692 mg, 1 mmol)in THF (9 mL) and methanol (1 mL), powdered KOH (168 mg, 3 eq) wasadded, and the solution stirred at r.t. for 2 hours. Saturated NH₄Cl (25mL) was added and the solution extracted 2×25 mL with ethyl acetate. Theorganic layer was dried over Na₂SO₄ and the solvent evaporated toprovide an off-white solid (650 mg, quantitative). ¹H NMR (CDCl₃) δ_(H)7.45-7.28 (20H, m, Ar—H), 7.08 (1H, s, H-2′), 6.99, 6.95 (2H, ABq, J=8.3Hz, H-5′, H-6′), 6.26, 6.15 (2×1H, 2×d, J=2.1 Hz, H-6, H-8), 5.16 (4H,s, CH₂Ph), 5.06 (3H, m, H-4, CH₂Ph), 4.97 (2H, s, CH₂Ph), 4.85 (1H, d,J=9.9Hz, H-2), 3.95(1H, m, H-3), 2.75 (1H, bs, OH), 2.55 (1H, bs, OH).

Example 9 Preparation of 4β-Hydroxyl tetra-O-benzyl-(−)-epicatechin

The title compound was prepared in an identical manner set forth inExample 8, except that 4β-acetoxy tetra-O-benzyl-(−)-epicatechin wasused in place of 4β-acetoxy tetra-O-benzyl-(+)-catechin to provide 287mg, 86% product. ¹H NMR (CDCl₃) δ_(H) 7.45-7.31 (20H, m, Ar—H), 7.16(1H, s, H-2′), 6.99, 6.95 (2H, ABq, J=8.3 Hz, H-5′, H-6′), 6.29, 6.27(2×1H, 2×d, J=2.1 Hz, H-6, H-8), 5.18(4H, s, CH₂Ph), 5.18 (4H, s,CH₂Ph), 5.07 (3H, m, H-4, CH₂Ph), 5.01 (2H, s, CH₂Ph), 4.92(1H, s, H-2),3.98 (1H, dd, J=2.5, 5.7 Hz, H-3), 2.43 (1H, d, J=2.4 Hz, OH), 1.58 (1H,d, J=5.7 Hz, OH).

Example 10 Preparation of 4β-Methoxy tetra-O-benzyl-(+)-catechin

To a solution of 4-acetoxy tetra-O-benzyl-(+)-catechin (70 mg, 0.1 mmol)in methylene chloride (5 mL) and methanol (1 mL), LiBr (87 mg, 10 eq)was added and the solution refluxed for 4 hours. The mixture was thenpartitioned between methylene chloride and water (25 mL each). Theorganic layer was dried over Na₂SO₄, filtered and the solventevaporated. The residue was subjected to silica gel chromatography toproduce 54 mg, 80% of the title compound as pale yellow oil. ¹H NMR(CDCl₃) δ_(H) 7.42-7.27 (20H, m, Ar—H), 7.07 (1H, d, J=1.6 Hz, H-2′),6.96 (2H, m, H-5′, H-6′), 6.26(1H, d, J=2.2 Hz, H-6 or H-8), 6.15 (1H,d, J=2.2 Hz, H-8, H-6) 5.15 (4H, s, CH₂Ph), 5.02(2H, ABq, J=7.8 Hz,CH₂Ph), 4.97 (2H, s, CH₂Ph), 4.92 (1H, d, J=10.4 Hz, H-2), 4.72 (1H, d,J=3.5Hz, H-4), 3.85 (1H, dt, J=3.5, 9.2 Hz, H-3), 3.47 (3H, s, OCH₃).

Example 11 Preparation ofTetra-O-benzyl-(+)-catechin-(4α→8)-(−)-epicatechin

4β-Acetoxy tetra-O-benzyl-(+)-catechin (Example 6) (140 mg, 0.2 mmol),(−)-epicatechin (290 mg, 5 eq) and LiBr (87 mg, 5 eq) were dissolved ina mixture of THF and methylene chloride (5 mL each) and the solutionrefluxed for 24 hours after which the solution was partitioned betweenethyl acetate and water (40 mL each). The organic layer was dried overNa₂SO₄ followed by evaporation of the solvent. The residue wasresuspended in ethyl acetate and filtered to remove most of the(−)-epicatechin. The filtrate was evaporated and subjected to silica gelchromatography where methylene chloride:ethyl acetate (1:1, v/v) elutefurnished 116 mg, 62% dimer as an off-white powder. For the NMR spectrumthe Hs comprising the upper monomer of the dimer are designated A andthe Hs comprising the lower monomer of the dimer are designated B. ¹HNMR (CDCl₃:d₄-menthanol, 9:1) δ_(H) 7.36-7.23 (20H, m, Ar—H),7.02-6.74(5H, m, A-5′, A-6′, A-2′, B-2′, B-5′), 6.35 (1H, dd, J=1.7, 8.2Hz, B-6′), 6.18-6.16 (2H, ABq, J=2.2 Hz, A-6, A-8), 5.86 (1H, s, B-6),5.12 (5H, m, CH₂Ph, B-2), 4.90 (2H, s, CH₂Ph), 4.71 (1H, d, J=8.2 Hz,A-2), 4.59 (1H, d, J=10 Hz, CH₂Ph), 4.47 (1H, d, J=10 Hz, CH₂Ph), 4.29(1H, dd, J=8.3, 8.3 Hz, A4), 3.80 (1H, m, H-3), 2.71 (1H, d, J=16.6 Hz,B-4), 2.53 (1H, dd, J=4.4, 16.6 Hz, B4); ¹³C NMR δ156.5, 156.0, 154.6,154.0, 152.5, 151.6, 151.4, 151.2, 150.6, 147.0, 146.7, 141.7, 141.6,141.5, 139.8, 134.8, 134.4, 134.2, 133.9, 129.3, 128.7, 126.2, 126.1,126.0, 125.8, 125.5, 125.4, 125.2, 125.1, 125.0, 124.9, 119.8, 115.0,112.6, 111.2, 106.1, 104.5, 96.5, 94.9, 93.0, 92.8, 79.9, 70.5, 69.1,69.0, 67.8, 67.7, 63.9, 35.0, 25.5; IR (KBr, cm⁻¹) 3418, 3057, 3034,2918, 1609, 1510, 1446, 1371, 1260, 1202, 1097, 806, 731, 696; MS (FAB,m/z) 939.6 (M+H)⁺, 649.1, 607.0, 559.0, 459.8.

Example 12 Preparation of (+)-Catechin (4α→8)-(−)-epicatechin

Tetra-O-benzyl-(+)-catechin-(4α→8)-(−)-epicatechin prepared in Example11 (50 mg) was dissolved in methanol (10 mL) and degassed by blowingargon for 10 min. 30% Palladium-charcoal (30 mg) was added andhydrogenolysis conducted at 45 psi for 3 hours. The solution wasfiltered through Celite which was washed with methanol. The combinedfiltrate and washings were evaporated and the residue was dissolved inwater, then lyophilized to provide a quantitative yield of the dimer asan off-white solid. For the NMR spectrum the Hs comprising the uppermonomer of the dimer are designated A and the Hs comprising the lowermonomer of the dimer are designated B. ¹H NMR (CDCl₃:d₄-methanol, 9:1)δ_(H) 7.21 (1H, bs, A-2′), 7.04 (1H, bs, B-2′), 6.95-6.75 (2H, m, A-5′,B-5′), 6.62 (1H, m, A-6′), 6.45 (1H, m, B-6′), 6.20 (1H, m, B-6), 6.05(1H, m, B-6), 5.89 (2H, m, A-6, A-8), 4.98 (1H, m, B-2,), 4.85 (1H, m,B-2), 4.42-4.25 (3H, m, A-4, A-3, A-2), 3.05-2.62 (2H, m, B-4).

Example 13 Preparation of3-Acetyl-tetra-O-benzyl-(+)-catechin-(4α→8)-pentaacetyl-(−)-epicatechin

Tetra-O-benzyl-(+)-catechin-(4α→8)-epicatechin prepared in Example 11was acetylated with acetic anhydride in pyridine. 120 mg of tetraO-benzyl-(+)-catechin (4α→8)-(−)-epicatechin was dissolved in 2 mL ofdry pyridine and 500 μL of acetic anhydride added. The reaction mixturewas stirred under argon for 18 hours at r.t. The mixture was partitionedbetween ethyl acetate and 1N HCl (25 mL each). The organic layer waswashed with 25 mL water, followed by 25 mL 5% sodium bicarbonate,followed by 25 mL saturated NaCl and the organic phase dried overNa₂SO₄. The product was purified by chromatography to provide 140 mg ofthe title compound at a yield of 91%. For the NMR spectrum the Hscomprising the upper monomer of the dimer are designated A and the Hscomprising the lower monomer of the dimer are designated B. ¹H NMR(CDCl₃: d₄-methanol, 9:1) δ_(H) 7.40-7.29 (20H, m, Ar—H), 7.19-7.13 (5H,m, A-2′, A-6′, B-2′, B-5′, B-6′), 6.92 (1H, d, J=8.3 Hz, A-5′), 6.52(1H, s, B-6), 6.21, 6.18 (2×1H, 2×d, J=2.3 Hz, A-6, A-8), 5.67 (1H, t,J=9.6, Hz, H-3), 5.14(3H, s, CH₂Ph, B-3), 5.0 (2H, s, CH₂Ph,), 4.98 (2H,s, CH₂Ph,) 4.84 (1H, d, J=9.1Hz, A-3), 4.75(1H, d, J=10.1 Hz, A-2),4.58, 4.41 (2H, ABq, J=9.2 Hz, CH₂Ph), 2.64 (2H, m, B4), 2.29 (6H, s,COCH₃), 2.26 (3H, s, COCH₃), 2.26 (3H, s, COCH₃) 1.76 (3H, s, COCH₃),1.74 (3H, s, COCH₃), 1.64 (3H, s, COCH₃); ¹³C NMR 169.6, 168.2, 168.0,167.5, 158.0, 156.2, 153.2, 149.2, 148.7, 147.1, 146.2, 142.5, 142.0,137.0, 136.8, 136.5, 136.2, 130.0, 129.8, 128.3, 128.2, 127.7, 127.6,127.3, 127.1, 122.8, 121.6, 121.4, 114.9, 114.8, 110.2, 108.4, 106.0,95.0, 94.6, 80.0, 75.5, 73.4, 71.4, 71.0, 70.5, 69.7, 66.5, 35.0, 26.2,20.6, 20.5, 20.4, 20.3, 20.0; MS (FAB, m/z) 1192 (M+H)⁺, 1131, 1039,949, 841, 691.

Example 14 Preparation of 3-Acetyl-(+)-catechin(4α→8)-pentaacetyl-(−)-epicatechin

3-Acetyl-tetra-O-benzyl-(+)-catechin (4α→8)-pentaacetyl-(−)-epicatechinprepared in Example 13 was dissolved in degassed ethyl acetate-methanol(3 mL each) and hydrogenated with 30% palladium-charcoal at 45 psi for 4hours. The solution was filtered through Celite and the solventevaporated to provide a quantitative yield of the dimer as a pale yellowsolid. For the NMR specturm the Hs comprising the upper monomer of thedimer are designated A and the Hs comprising the lower monomer of thedimer are designated B. ¹H NMR (CDCl₃: d₄-methanol, 9:1) δ_(H) 7.47,6.98 (2×1H, bs, B-2′), 7.36, 6.98 (2×1H, 2×d, J=8.4, B-6′), 7.24, 7.16(2×1H, 2×d, J=8.4Hz, B-5′), 6.89, 66.0(2×1H, 2×bs, A-2′), 6.83, 6.79(2×1H, ABq, J=8.4 Hz, A-5′, A-6′), 6.66 (1H, d, J=8.4Hz, A-5′), 6.47(1H, d, J=8.4 Hz, A-6′), 6.51, 6.45 (2×1H, 2×s, B-6), 5.97, 5.84 (2×1H,2×d, J=2 Hz, A-6, A-8), 5.96 (2H, s, A-6, A-8), 5.71 (2H, m, A-3), 5.50,5.17 (2×1H, 2×bs, B-3), 5.28 (2×1H, 2×bs, B-2), 5.0, 4.54 (2×1H, 2×d,J=8.9, 9.4 Hz, A-2), 4.74, 4.61 (2×1H, 2×d, J=9.9 Hz, A-4), 3.2,2.74(2×2H, 2×m, B-4), 2.32, 2.29, 2.28, 2.26, 2.23, 2.01, 1.80, 1.77, 1.63(s, COCH₃); ¹³C NMR 172.5, 172.0, 171.8, 171.5, 170.3, 170.2, 170.1,170.0, 169.8, 169.2, 157.2, 157.1, 156.8, 154.8, 154.0, 149.2, 147.5,146.0, 145.9, 145.6, 145.0, 143.0, 142.8, 137.5, 137.0, 129.8, 125.8,124.9, 124.0, 123.0, 122.6, 121.8, 120.8, 120.2, 119.8, 115.9, 115.8,115.5, 115.1, 110.8, 110.9, 109.5, 109.4, 105.0, 104.2, 98.0, 97.5,96.8, 96.0, 81.0, 80.2, 79.0, 78.8, 78.6, 78.0, 75.0, 72.2, 68.1, 68.0,37.5, 36.0, 26.5, 26.0, 20.7.

Example 15 Preparation ofTetra-O-benzyl-(−)-epicatechin-(4β→8)-(−)-epicatechin

4β-Acetoxy tetra-O-benzyl-(−)-epicatechin prepared by Example 7(70 mg.0.1 mmol), (−)-epicatehin (145 mg, 5 eq) and LiBr (44 mg, 5 eq) weredisolved in a mixture of THF and methylene chloride (3 mL each) and thesolution refluxed for 24 hours under argon. The solution was partitionedbetween ethyl acetate and water (25 ml each) and the organic layer driedover Na₂SO₄. The solvent was evaporated and the residue resuspended in25 ml ethyl acetate followed by filtration to remove most of theunreacted (−)-epicatechin. The filtrate was evaporated to a residue andthe title compound isolated from silica gel chromatography by methylenechloride:ethyl acetate (1:1, v/v) elution. Evaporation of the eluateprovided 56 mg (60%) of an off-white powder. ₁NMR (CDCl₃:d₄menthanol,9:1) δ_(H)[No assignment]7.35-7.14 (20H, m), 6.92 (1H, bs), 6.82 (1H,s), 6.29 (1H, s) 6.18 (1H, s), 5.85 (1H, s), 5.01 (4H, s), 4.94 (2H, s),4.93 (2H, s), 4.38 (1H, s), 3.93 (1H, s,) 2.85 (2H, s).

Example 16 Preparation of (−)-Epicatechin-(4β→8)-(−)-epicatcehin

Tetra-O-(−)epicatechin (4β→8)-(−)-epicatcchin prepared in Example 15 (40mg, 0.043 mmol) was dissolved in 8 mL methanol and degassed by blowingargon for 10 min. To the solution, 25 mg of 30% palladium-charcoal wasadded and the mixture hydrogenolyzed at 45 psi for 3 hours. The solutionwas filtered through Celite followed by washing with 25 mL methanol. Thecombined filtrate and washing were evaporated and the residue dissolvedin water. Lyophilization provided 23 mg of an off-white powder. HPLCanalysis revealed the presence of 18% monomer, 45% dimer, 25% trimer and8% tetramer.

Example 17 Preparation ofTetra-O-benzyl-(+)-catechin-(4α→8)-(+)-catechin

4β-Acetoxy tetra-O-benzyl-(+)-catechin prepared by Example 6 (70 mg. 0.1mmol), (+)-catechin (145 mg, 5 eq) and LiBr (44 mg, 5 eq) were dissolvedin a mixture of THF and methylene chloride (3 mL each) and the solutionrefluxed for 24 hours under argon. The solution was partitioned betweenethyl acetate and water (25 mL each) and the organic phase dried overNa₂SO₄. Following evaporation, the residue was resuspended in ethylacetate (25 mL) and filtered to remove most of the unreacted(+)-catechin. After evaporation, the residue was subjected to silica gelchromatography where elution with methylene chloride:ethyl acetate (1:1,v/v) provided an off-white powder (81 mg, 68%) after evaporation. ¹H NMR(CDCl₃:d₄-methanol, 9:1) δ_(H) 7.39-7.06 (20H, m), 6.84-6.68 (5H, m),6.47(1H, d, J=7.9 Hz), 6.32-5.98 (4H, m), 5.00-4.33 (11H, m), 3.58 (1H,m), 2.98 (1H, m), 2.35 (1H, m); IR (KBr, cm⁻¹) 3441, 3057, 3034, 2918,1609, 1542, 1510, 1371, 1266, 1097, 812, 737, 696; MS (APCI, m/z) 938(M−H), 920, 848, 816, 696, 607, 558.

Example 18 Preparation of3-Acetyl-tetra-O-benzyl-(+)-catechin-(4α→8)-3-acetyl tetraO-benzyl-(−)-epicatechin

3-Acetyl tetra-O-benzyl-(+)-catechin (4α→8)-pentaacetyl-(−)-epicatechinprepared by Example 13 (119 mg, 0.1 mmol) was added to dry DMF (4 mL) at0° C., followed by NaH (29 mg, 4.2 eq), followed by benzyl bromide (54μL, 4.5 eq). Water (8 μL, 4 eq) was added slowly over 2 minutes and themixture was stirred for 24 hours at r.t. The solution was partitionedbetween ethyl acetate and water (25 mL each) and the organic phase wasdried over MgSO₄. Following evaporation, the residue was subjected tosilica gel chromatography where elution with 20% ethyl acetate in hexaneprovided 105 mg (90%) of the title compound after evaporation. For theNMR spectrum the Hs comprising the upper monomer of the dimer aredesignated A and the Hs comprising the lower monomer of the dimer aredesignated B. ¹H NMR (CDCl₃ d₄-methanol, 9:1) δ_(H) 7.45-7.24 (40H, m,Ar—H), 6.90-6.78 (6H, m, A-2′, A-5′, A-6′, B-2′, B-5′, B-6′), 6.22 (2H,m, A-6, A-8), 6.21 (1H, s, B-6), 5.92 (1H, m, A-3), 5.21-4.40 (20H,complex, CH₂Ph, A-2, A-4, B-2, B-3), 2.70 (2H, m, B-4), 1.67 (6H, s,CH₂Ph,), COCH₃).

Example 19 Preparation ofTetra-O-benzyl-(+)-catechin-(4α→8)-(+)-tetra-O-benzyl-(−)-epicatechin

To a solution of 3-acetyl tetra-O-benzyl-(+)-catechin-(4α→8)-3 acetyltetra-O-benzyl-(−)-epicatechin prepared in Example 18 (40 mg, 0.03 mmol)in the THF (2 mL) and methanol (200 μL), powdered KOH (5 mg, 3 eq) wasadded and the solution stirred at r.t. for 18 hours under argon. Thereaction mixture was partitioned between ethyl acetate and saturatedNH₄Cl (25 mL each). The organic layer was dried over MgSO₄ and thesolvent evaporated. The residue was then subjected to silica gelchromatography where elution with 20% ethyl acetate in methylenechloride provided the title compound (31 mg, 82.5%) as a colorless oilafter evaporation of the solvent. For the NMR spectrum the Hs comprisingthe upper monomer of the dimer are designated A and the Hs comprisingthe lower monomer of the dimer are designated B. ¹H NMR (CDCl₃d₄-methanol, 9:1) δ_(H) 7.41-7.13 (40H, m, Ar—H), 6.97-6.79 (6H,complex, A-2′, A-5′, A-6′, B-2′, B-5′, B-6′), 6.22 (1H, s, B-6), 6.20,6.12 (2×1H, 2×d, J=2.4 Hz, A-6, A-8), 5.18-4.51 (19H, CH₂Ph, A-2, A-4,-2), 4.28 (1H, m, A-3), 3.85 (1H, m, B-3), 2.95 (1H, d, J=16 Hz, B4),2.60 (1H, dd, J=5, 16 Hz, B4).

Example 20 Preparation of Tetra-O-benzyl-(+)catechin-(4α→8)-3-acetyl-(+)-catechin-(4α→8)-pentaacetyl (−)-epicatechin

To a solution of 3-acetyl-(+)-catechin-(4α→)-pentaacetyl-(−)-epicatechinprepared by Example 14 (100 mg, 0.068 mmol) and 4βacetoxytetra-O-benzyl-(+)-catechin prepared by Example 6 (334 mg, 2 eq) in THF(7 mL) and methylene chloride (7 mL), 161 mg of LiI was added. Thesolution was refluxed for 24 hours, followed by partition between ethylacetate and water (25 mL each). The organic phase was dried over Na₂SO₄,filtered and the solvent evaporated. The residue was subjected to silicagel chromatography where elution with ethyl acetate-niethylene chloride(1:1, v/v) provided a brownish white solid (100 mg, 28%) after theevaporation of the solvent. MS (FAB, m/z) 1482 (M+H)⁺, 1148, 1042, 962,920, 650.

Example 21 Preparation of Tetra-O-benzyl-(+)-catechin-(4α→8)-pentaacetyl-(+)-epicatechin-(4α→8)-pentaacetyl (−)-epicatechin

Tetra-O-benzyl-(+)catechin-(4α→8)-3-acetyl-(+)-catechin-(4α→8)-pentaacetyl (−)-epicatechinprepared by Example 20 (100 mg, 0.068 mmol) was stirred in dry pyridine(2 mL) and acetic anhydride (1 ml) under argon for 24 hours. Thesolution was then partitioned between 1 N HCl and ethyl acetate (25 mLeach), the organic layer was washed with 5% NaHCO₃, saturated NaCl anddried over MgSO₄. Evaporation of the solvent provided an oily residuewhich was subjected to silica gel chromatography where elation with 10%ethyl acetate in methylene chloride provided a white powder (70 mg, 61%)after evaporation of the solvent.

Example 22 Preparation of 3 Acetyl-(+)-catechin -(4α→8)-pentaacetyl-(+)-catechin-(4α→8)-pentaacetyl (−)-epicatechin

Tetra-O-benzyl-(+)-catechin-(4α→8)-pentaacetyl-(+)-catechin-(4α→8)-pentaacetyl(−)-epicatechin prepared in Example 21 (50 mg) was dissolved in degassedethyl acetate-methanol (3 mL each) and hydrogenolysed for 4 hours in thepresence of 30% palladium-charcoal (30 mg) at 45 psi. Removal of thecatalyst via filtration through Celite and evaporation provided thetitle compound as a pale brown powder (35 mg, 91%).

Example 23Tetra-O-benzyl-(+)-catechin-(4α→8)-3-acetyl-(+)-catechin-(4α→8)-pentaacetyl-(+)-catechin-(4α→8)-pentaacetyl(−)-epicatechin

To a solution of3-acetyl-(+)-catechin-(4α→8)-pentaacetyl-(+)-catechin-(4α→8)-pentaacetyl(−)-epicatechin prepared by Example 22 (30 mg, 0.0226 mmol) and4β-acetoxy tetra-O-benzyl-(+)-catechin prepared by Example 6 (31 mg, 2eq) in THF and methylene chloride (2 mL each), LiI (16 mg, 5 eq) wasadded and the solution refluxed for 24 hours. The solution waspartitioned between ethyl acetate and water (25 mL each) and the organiclayer dried over MgSO₄, filtered and the solvent evaporated. The residuewas subjected to silica gel chromatography where elution with 10%methanol in methylene chloride provided a brownish-white solid (20 mg,45%) after evaporation of the solvent. MS (FAB, m/z) 1978 (M+H)⁺, 1934(M⁺-COCH₃), 1571 (M⁺-COCH₃, −3×CH₂Ph), 1646, 1430, 1373, 1330, 1269.

Example 24 Preparation ofTetra-O-benzyl-(+)-catechin-(4α→8)-(−)-epicatechin-(6→4α)-tetra-O-benzyl-(+)-catechin

To a solution of tetra-O-benzyl-(+)-catechin-(4α→8)-(−)-epicatechinprepared by Example 11 (69 mg, 0.074 mmol) and 4β-acetoxytetra-O-benzyl-(+)-catechin prepared by Example 6 (51 mg, 0.074 mmol) inmethylene chloride and THF (5 mL each), LiBr (65 mg, 10 eq) was addedand the mixture refluxed for 24 hours. The solution was partitionedbetween ethyl acetate and water (25 mL each) and the organic layer driedover MgSO₄. The solvent was evaporated and the residue subjected tosilica gel chromatography where elution with ethyl acetate-methylenechloride (1:1, v/v) provided a

white powder (35 mg, 30%) after evaporation of the solvent. MS (FAB,m/z) 1588 (M+H)⁺, 1255, 772, 648, 607, 560.

Example 25 Preparation of 8-Bromo tetra-O-benzyl-(−)-epicatechin

To a solution of tetra-O-benzyl-(−)-epicatechin (Example 2) (65 mg, 0.1mmol) in methylene chloride (2 mL), N-bromosuccinimide (18 mg, 0.1 mmol)was added and the solution stirred under argon for 10 min. The mixturewas filtered through silica gel followed by elution with 20 mL ethylacetate:methylene chloride (1:1, v/v). The combined filtrate and eluantwere evaporated. The residue was subjected to silica gel chromatographywhere elution with methylene chloride provided the title compound asshiny pinkish-white crystals (66 mg, 90%) after evaporation of thesolvent. ¹H NMR (CDCl₃) δ_(H), 7.45-7.21 (21H, m, Ar—H), 7.01 (1H, dd,J=1.4 8.3 Hz, H-6′), 6.96(1H, d, J=8.3Hz, H-5′), 6.23 (2H, s, H-6), 5.38(1H, m, H-3), 5.21, 5.18, 5.10, 4.97 (4×2H, 4×s, 4×CH₂), 5.01(1H, s,H-2), 4.3 (1H, m, H-3), 3.03 (1H, dd, J=1.9, 17.4 Hz, H-4), 2.89 (1H,dd, J=4., 17.4Hz, H-4), 1.55 (1H, d, J=4.8 Hz, OH).

Example 26 Preparation of 8-Bromo penta-O-benzyl-(−)-epicatechin

To a solution of pentabenzyl-(−)-epicatechin (55 mg, 0.074 mmol) inmethylene chloride (2 mL) at 0° C., N-bromosuccinamide (14 mg, 1 eq) wasadded and the solution stirred at r.t. for 30 min. The solution waspassed through a 25 mm dia. column of silica gel (7 gm) which was elutedwith methylene chloride (30 mL). The combined filtrate and eluant wereevaporated to provide the title compound as a white foam (50 mg, 82.5%)after evaporation of the solvent. ¹H NMR (CDCl₃) δ_(H), 7.43-6.90 (28H,m, Ar—H), 6.21 (1H, s, H-6), 5.17 (2H, s, CH₂), 5.09 (5H, s, 2×CH₂,H-2), 4.96(2, s, CH₂), 4.37, 4.27 (2H, AB, J=12.6 Hz, 3-OCH₂), 3.95 (1H,m, H-3), 2.94 (1H, dd, J=3.6, 17.1 Hz, H-4), 2.78 (1H, dd, J=4.4, 17.1Hz, H-4).

Example 27 Preparation of 8-bromo 4β-acetoxy pentabenzyl-(−)-epicatechin

To a mixture of 8-bromo pentabenzyl-(−)-epicatechin (Example 26) (59 mg,0.072 mmol) and lead tetraacetate (48 mg, 1.5 eq) under argon, benzene(2 mL) was added, followed by 2 mL acetic acid, and the mixture stirredfor 60 hours at r.t. The solution was partitioned between ethyl acetateand water (50 mL each). The organic layer was washed with 1N NaOH (2×50mL), followed by water (50 mL), saturated NaCl (50 mL), and was driedover Na₂SO₄. The solution was filtered and evaporated to provide an oilyresidue which was subjected to silica gel chromatography where elutionwith 20% ethyl acetate in hexane provided the title compound as a whitefoam (38 mg, 60%) after evaporation. ¹H NMR (CDCl₃) δ_(H) 7.46-6.85(28H, m, Ar—H), 6.25 (1H, s, H-6), 6.18(1H, d, J=2.3 Hz, H-4), 5.20,5.13, 5.02 (4×2H, 4×s, 4×CH₂), 4.99 (1H, s, H-2), 4.51, 4.33 (2H, AB,J=12.3) Hz, 3-OCH₃), 3.65 (1H, m, H-3), 2.0 (2H, s, OCOCH₃).

Example 28 Determination of Absolute Configuration 8-bromotetra-O-benzyl-(−)-epicatechin

Crystals of 8-bromo tetra-O-benzyl-(−)-epicatechin were mounted on glassfibers and placed in a cold N₂ stream at −44° C. on a Siemens SMART CCDX-ray diffractometer. In general, the crystals diffracted poorly, withfew or no high-angle reflections and with weak intensities overall. Thefirst three crystals did not diffract well enough to measure the unitcell even at longer than usual exposure times. The fourth diffractedwell enough to refine a unit cell using fifteen reflections. Data werecollected on this crystal using fifty second exposures for over twothousand frames to approximately cover the diffraction sphere using Moradiation.

The unit cell volume indicated two molecules in the unit cell. Althoughone cell angle was clearly 90° and one was clearly different from 90°(92.6°), the third angle differed from 90° by 0.1° which is a largererror than usual for a monoclinic cell. However, examination of possiblesystematic absences showed an apparent 2₁ axis consistent with themonoclinic space group P2₁ appropriate for a chiral compound. Subsequentsuccessful structure solution and refinement in P2₁ supported thatchoice. The structure was solved by direct methods using the SHELXpackage (Sheldrick, G. M. SHELXTL Structure Determination SoftwarePrograms: Siemens Analytical X-ray Instruments Inc. Madison, Wis., 1990)of programs. Hydrogen atoms were placed in fixed, calculated positions.Phenyl rings in benzyl groups were refined isotropically as rigidgroups. No corrections were made for absorption or extinction. Thefollowing table lists the crystal data.

TABLE Crystal Data and Structure Refinement for 8-bromotetra-O-benzyl-(−)-epicatechin Empirical Formula C₄₃H₃₇BrO₆ FormulaWeight 729.64 Temperature 229(2)° K Wavelength 0.71073 A° Crystal SystemMonolinic Space Group P2₁ Unit Cell Dimensions a = 15.9122(8) A° alpha =90° b = 4.8125(3) A° beta = 92.6390(10)° c = 22.4772(13) A° gamma = 90°Volume, z 1719.42(17) A°³, 2 Density (calculated) 1.409 Mg/m³ Absorptioncoefficient 1.246 mm⁻¹ F(000) 756 Crystal Size 0.45 × 0.04 × 0.04 mm θrange for data collection 1.28 to 23.44° Limiting Indices −17 ≦ h ≦ 17,−5 ≦ k ≦ 5, −24 ≦ l ≦ 24 Reflections collected 13371 Independentreflections 4868 (R_(int) = 0.2246) Completeness to θ = 23.44° 99.2%Absorption correction None Refinement method Full matrix least squareson F² Data/Restraints/Parameters 4868/1/274 Goodness of fit on F² 1,091R indices (all) R1 = 0.2263, wR2 = 0.4749 Final R indices [I > 2σ (I)]R1 = 0.1515, wR2 = 0.3875 Absolute structure 0.00(5) parameter Largestdiff. peak and hole 1.857 and −1,268 eA°³

The assignment of the correct absolute configuration was tested bycalculation of the Flack ‘x’ parameter. This parameter wasindistinguishable from zero, indicating the correct configuration wasassigned. A test refinement of the inverted configuration resulted in aFlack ‘x’ parameter value of 0.95(5) and a significant increase in the Rfactors, both indicating that the assignment was correct.

Example 29 Preparation of (88), (86), and (66) Linked ProcyanidinOligomers

The steps described in this invention can be extended to provideprocyanidin oligomers comprising (8

8), (8

6), (6

6) interflavan linkages. These compounds are obtained from 6-bromo-and/or 8-bromo-(monomer) intermediates. Coupling of these brominatedmonomers with organotin derivatives by a Stille reaction in the presenceof a palladium_((o)) catalyst leads to the desired oligomeric linkage.(Stille, J. K., Agnew, Chem. Internal. Ed. Engl., 25, 508-524 (1986)).

For instance, 8-bromo pentabenzyl-(−)-epicatechin prepared by Example 26reacted with hexaabutyl distannane to provide the alkyl stannane ofpentabenzyl (−)-epicatechin. Coupling of this stannnane with another8-bromo pentabenzyl (−)-epicatechin in the presence of tetrakis(triphenyl phosphine) palladium₍₀₎ in benzene provides thedecabenzyl-(−)-epicatechin dimer with an (8→8) linkage. Deprotectingwith H₂/Pd provides the (−)-epicatechin-(8

8)-(−) -epicatechin in free phenolic form.

Similarly, procyanidin oligomers comprising (8

6) or (6

6) linkages can be synthesized using the appropriate 6-bromo- or8-bromo-(monomer) derivatives. Further, coupling of 8-bromo- or 6-bromo-dimers, trimers and higher oligomers can provide “even” numberedprocyanidin oligomers comprising (8

8), (8

6), and (6

6) linkages.

Still further, coupling of blocked monomers used to prepare (4→6) linkedoligomers as described in the invention can be used in the Stillereaction to provide novel procyanidin oligomers comprising combinationsof the (4→6) and (4→8) linked oligomers with (8

8), (8

6), and (6

6) linkages. By way of example, the following structure illustrates and(8

8) and (4

8) linked procyanidin trimer.

1. A process for preparing a partially protected procyanidin dimer whichcomprises the steps of: (a) protecting each phenolic hydroxyl group ofan epicatechin or a catechin monomer; (b) activating the monomer fromstep (a) by introducing an acyloxy group at the C-4 position; and (c)catalytically coupling the monomer from step (b) with an unprotectedepicatechin or an unprotected catechin monomer to form a partiallyprotected procyanidin dimer where the top mer is protected and where thebottom mer is unprotected.
 2. The process of claim 1, wherein theprotecting groups are benzyl groups and wherein the protecting step iscarried out with benzyl bromide in dimethyl formamide or dimethylacetamide.
 3. The process of claim 1, wherein the protecting groups arep-methoxybenzyl groups and the protecting step is carried out withp-methoxybenzyl chloride in dimethyl formamide.
 4. The process of claim2, wherein the protecting step is carried out in the presence ofpotassium carbonate or potassium iodide.
 5. The process of claim 1,wherein the activating step is carried out using as an oxidizing agent alead salt selected from the group consisting of lead acetate, leadformate, and lead propionate in a solvent selected from the groupconsisting of benzene, toluene, chlorobenzene, cyclohexane, heptane,carbon tetrachloride, and mixtures thereof.
 6. The process of claim 5,wherein the lead acetate is used in combination with acetic acid;wherein the lead formate is used in combination with formic acid;wherein the lead propionate is used in combination with propionic acid;and wherein the solvent in the activating step is benzene-acetic acid.7. The process of claim 1, wherein the catalyst used in the couplingstep is a Lewis acid selected from the group consisting of lithiumbromide and lithium iodide.
 8. A process for preparing a linearprocyanidin oligomer having 4→8 linkages, which process comprises thesteps of: (a) preparing a partially protected 4→8 procyanidin dimerwhere the phenolic hydroxyl groups of the top mer are protected withremovable protecting groups which do not deactivate the protected merand where the phenolic hydroxyl groups of the bottom are unprotected;(b) masking the dimer of step (a) with removable masking groups whichdeactivate the bottom mer to form a dimer where the phenolic hydroxylgroups of the top mer are protected, where the phenolic hydroxyl groupsof the bottom mer are masked, and where the hydroxyl groups at the C-3positions of both mers are masked; (c) deprotecting the dimer of step(b) to form a dimer where the phenolic hydroxyl groups of the top merare unprotected, where the phenolic hydroxyl groups of the bottom merare masked, and where the hydroxyl groups at the C-3 positions of bothmers are masked; (d) catalytically coupling the dimer of step (c) with aprotected catechin monomer or a protected epicatechin monomer having anacyloxy activating group at the C-4 position to form a 4→8 trimer wherethe phenolic hydroxyl groups of the top mer are protected, where thephenolic hydroxyl groups of the middle mer are unprotected, where thephenolic hydroxyl groups of the bottom mer are masked, and where thehydroxyl groups at C-3 positions of the middle and the bottom mers aremasked.
 9. The process of claim 8, further comprising the step(s) ofdemasking and/or deprotecting the trimer of step (d).
 10. The process ofclaim 8, which process further comprises the steps of: (e) masking thetrimer of step (d) to form a trimer where the phenolic hydroxyl groupsof the top mer are protected, where the phenolic hydroxyl groups of themiddle and bottom mers are masked, and where the hydroxyl groups at theC-3 positions of all the mers are masked; (f) deprotecting the trimer ofstep (e) to form a trimer where the phenolic hydroxyl groups of the topmer are unprotected, where the phenolic hydroxyl groups of the middleand bottom mers are masked, and where the hydroxyl groups at the C-3positions of all the mers are masked; and (g) catalytically coupling thetrimer of step (f) with a protected catechin monomer or a protectedepicatechin monomer having an acyloxy activating group at the C-4position to form a 4→8 tetramer where the phenolic hydroxyl groups ofthe top mer are protected, where the phenolic hydroxyl groups of themiddle mers and the bottom mer are masked, and where the hydroxyl groupsat the C-3 positions of the middle mers and the bottom mer are masked;and (h) optionally repeating the masking, deprotecting, and couplingsteps to form a higher oligomer.
 11. The process of claim 10, furthercomprising the step(s) of demasking and/or deprotecting the tetramer ofstep (g) or higher oligomer of step (h).
 12. The process of claim 10,wherein the higher oligomers include pentamers through dodecamers. 13.The process of claim 10, wherein the higher oligomers are the pentamers.